, 120133, published online 31 October 2012 2 2012 Open Biol. Zhenxun Wang, Hyun Yong Jeon, Frank Rigo, C. Frank Bennett and Adrian R. Krainer by antisense oligonucleotides Manipulation of PK-M mutually exclusive alternative splicing Supplementary data http://rsob.royalsocietypublishing.org/content/suppl/2012/10/30/rsob.120133.DC1.html "Data Supplement" References http://rsob.royalsocietypublishing.org/content/2/10/120133.full.html#ref-list-1 This article cites 31 articles, 13 of which can be accessed free any medium, provided the original work is properly cited. Attribution License, which permits unrestricted use, distribution, and reproduction in This is an open-access article distributed under the terms of the Creative Commons Subject collections (27 articles) molecular biology (3 articles) biotechnology (29 articles) biochemistry Articles on similar topics can be found in the following collections Email alerting service here right-hand corner of the article or click Receive free email alerts when new articles cite this article - sign up in the box at the top on January 17, 2013 rsob.royalsocietypublishing.org Downloaded from
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, 120133, published online 31 October 20122 2012 Open Biol. Zhenxun Wang, Hyun Yong Jeon, Frank Rigo, C. Frank Bennett and Adrian R. Krainer by antisense oligonucleotidesManipulation of PK-M mutually exclusive alternative splicing
This article cites 31 articles, 13 of which can be accessed free
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Manipulation of PK-M mutuallyexclusive alternative splicing byantisense oligonucleotidesZhenxun Wang1,2,†, Hyun Yong Jeon1,3, Frank Rigo4,
C. Frank Bennett4 and Adrian R. Krainer1,2
1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA2Watson School of Biological Sciences, Cold Spring Harbor, NY 11724, USA3Graduate Program in Molecular and Cellular Biology, Stony Brook University,Stony Brook, NY 11794, USA4Isis Pharmaceuticals, Carlsbad, CA 92008, USA
1. SummaryAlternative splicing of the pyruvate kinase M gene involves a choice between
mutually exclusive exons 9 and 10. Use of exon 10 to generate the M2 isoform
is crucial for aerobic glycolysis (the Warburg effect) and tumour growth. We pre-
viously demonstrated that splicing enhancer elements that activate exon 10 are
mainly found in exon 10 itself, and deleting or mutating these elements increases
the inclusion of exon 9 in cancer cells. To systematically search for new enhancer
elements in exon 10 and develop an effective pharmacological method to force a
switch from PK-M2 to PK-M1, we carried out an antisense oligonucleotide
(ASO) screen. We found potent ASOs that target a novel enhancer in exon 10
and strongly switch the splicing of endogenous PK-M transcripts to include
exon 9. We further show that the ASO-mediated switch in alternative splicing
leads to apoptosis in glioblastoma cell lines, and this is caused by the downregula-
tion of PK-M2, and not by the upregulation of PK-M1. These data highlight the
potential of ASO-mediated inhibition of PK-M2 splicing as therapy for cancer.
2. IntroductionCancer cells preferentially use the glycolytic metabolic pathway with lactate
generation, even under normal oxygen conditions [1]. This metabolic feature
is termed the Warburg effect. Expression of the type II isoform of the
pyruvate kinase M gene (PKM2, referred to here as PK-M) has been shown to
mediate this effect, and to facilitate the proliferation of cancer cells in vivo [2].
The PK-M gene consists of 12 exons; exons 9 and 10 are alternatively spliced
in a mutually exclusive fashion to give rise to M1 and M2 isoforms, respectively
[3]. PK-M catalyses the final step in glycolysis to generate pyruvate and ATP
from phosphoenolpyruvate and ADP [4]. Exons 9 and 10 each encode a
56-amino acid segment that confers distinctive properties to the respective
PK-M isozymes. PK-M1 is constitutively active, whereas PK-M2 is allosteri-
cally regulated by fructose-1,6-bisphosphate levels and interaction with
tyrosine-phosphorylated signalling proteins [5].
Consistent with the correlation between proliferation and PK-M2
expression, PK-M2 is highly expressed in embryonic tissues and in a broad
range of cancer cells, whereas PK-M1 is predominantly expressed in terminally
differentiated tissues [2,6]. In particular, the mammalian target of rapamycin
pathway, which is a central mediator of cellular growth and proliferation,
Figure 1. Antisense oligonucleotide (ASO) walk along the 10W region. (a) Diagram of the PK-M genomic region, and the RT-PCR assay to measure M1/M2 ratios. Thisregion comprises introns 8, 9 and 10 (represented by the lines), intact exon 9 (green box), exon 10 (red box) and portions of exons 8 (white box) and 11 (black box).Numbers above the boxes show the length in nucleotides. Primers annealing to exons 8 and 11 used to amplify the endogenous PK-M transcript are represented by thearrows. cDNA amplicons generated after radioactive PCR are shown below and labelled accordingly. Three spliced species were observed: the shorter double-skipped species,comprising only exons 8 and 11 (D, 271 nt); M1, including exon 9 (A, 398 nt); and M2, including exon 10 (B, 398 nt). To distinguish between M1 and M2, a subsequentPstI digest was carried out. Only M2 has a PstI site, resulting in two cleavage products: B1 (213 nt) and B2 (185 nt), which are the 30 and 50 ends of M2, respectively.(b) Scheme of the ASO screen focused on the 10W region. The sequence of exon 10 from 25 to 88 nt upstream of the 50 splice site (ss) is indicated in red. Stacked linesrepresent individual ASOs and are aligned to the complementary sequence in exon 10. The ASO names are indicated on the left, with the subscript numbers indicating thetarget-sequence coordinates. The initial ASO walk is represented at the top, with ASO 10W45 – 59 indicated in red and shown to be annealing to the 10W region (boundedby the rectangle) by vertical dashed lines. The microwalk ASOs are indicated below, and the complementary sequence targeted by ASO 10M46 – 60 is indicated with verticaldashed lines. (c,d ) ASO walks. Radioactive RT-PCR and restriction digest of endogenous PK-M transcripts in HEK-293 cells after transfection of ASOs. Initial walk ASOs,transfected at 30 nM, are shown in (c), and microwalk ASOs transfected at 60 nm are shown in (d ). RNAs were harvested from cells 48 h after transfection. The transfectedASO is indicated at the top. cDNA amplicons and fragments are indicated on the left. Lane numbers and quantifications are indicated at the bottom. Each product wasquantified as a percentage of the total of M1, M2 and double-skipped species. %M1 and %M2 are shown. All standard deviations are �4% (n¼ 3).
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10W45–59 10M46–60 ASO: control 10W45–59 10M46–60 10W45–59 10M46–60
exon 10 to exon 9 subexonic duplication
duplicatedregion:
10W
10F
10B
regiontargeted by:
ASO
10W45–59
M1
M2
Ex 10 10W region
10
10W10F10B 10M46–60
G7 ASO 3¢ -5¢
Figure 2. Characterizing the 10W ASO target region. (a) Scheme of method used to duplicate the exon 10 10W region into exon 9 in a minigene. The minigene comprises thesame genomic region as indicated in figure 1a. To amplify minigene transcripts, we used a primer annealing to a vector-specific sequence upstream of the genomic insert, pcDNAF[14]. Minigene mutant names are indicated below. The indicated exon 9 (green) nucleotides at the top were mutated to the corresponding exon 10 (red) sequences on the right.The 10W minigene duplicates the entire exon 10 10W region into exon 9; the 10F minigene duplicates the first 8 nt of 10W45 – 59; and the 10B minigene duplicates the last 7 ntof 10W45 – 59. The ASOs that target 10W and the flanking regions are indicated below. (b) The 10W region is an exon 10 ESE. Mutant minigenes were analysed by transienttransfection into HEK-293 cells, followed by radioactive RT-PCR and restriction digestion, as in figure 1. Constructs from (a) are labelled at the top. Labelled bands are indicated inlower case on the left and right, with important bands in blue font. %M1 is indicated at the bottom. Bands are as follows: uncut M1 fragment (a, 481 nt); uncut M2 fragment(b, 481 nt); PstI-cleaved M2 50 fragment (b2, 268 nt); PstI-cleaved M2 30 fragment (b3, 213 nt); a spliced mRNA that skips both exons 9 and 10 (d, 314 nt); an exon 9 – exon10 doubly included mRNA expressed from the 10B minigene (lanes 5 and 6) is indicated on the left (f, 648 nt). This band is sensitive to PstI (f1, 435 nt). Standard deviations (s.d.)are 0.2%, 0.3% and 2.6% for 10G, 10F and 10B, respectively; n¼ 3. (c,d ) Minigene transcript-level changes as a result of ASO co-transfection in HEK-293 cells. ASOs weretransfected at a nominal final concentration of 60 nM. The wild-type (c) and exon 10 duplication (d ) minigenes [14], together with the identity of the ASOs, are indicated at thetop. Labelled bands are indicated in lower case on the left, with important bands in blue font. The exon 10 – exon 10 doubly included mRNA in (d ) expressed from the exon 10duplication minigene is indicated on the right (g, 648 nt). %M1, %M2 or %Skp is indicated at the bottom. Standard deviations for (c) are 0.6%, 4.2% and 2.9% for control,10W45 – 59 and 10M46 – 60, respectively; s.d. for (d ) are 0.8%, 9.4% and 2.6% for control, 10W45 – 59 and 10M46 – 60, respectively; n¼ 3. (e) Diagram of a homologous potentialcross-hybridizing region for ASOs 10W45 – 59 and 10M46 – 60 ASOs. Alignment of the sequences of ASO 10W45 – 59, the complementary region in exon 10 (indicated in red), and ahomologous region in intron 9 (indicated in blue, 129 – 156 nt upstream of the exon 9 50ss) is shown. Vertical lines indicate sequence identity. Diagram of minigene mutants usedin ( f – h) is shown on the left. d10W has the 10W ASO binding site in exon 10 removed and replaced by the corresponding region in exon 9. dInt9 has a 15 nt deletion of thehomologous intron 9 region (e). dM has both mutations. ( f – h) Minigene transcript-level changes as a result of ASO co-transfection in HEK-293 cells. ASOs were transfected at afinal nominal concentration of 60 nM. The minigenes, together with the identity of ASOs, are indicated at the top. Labelled bands are indicated in lower case on the left, withimportant bands in blue font. ASOs and minigenes from (e) are indicated at the top, %M1 is indicated at the bottom and bands are indicated on the left. %M1, %M2 or %Skp isindicated at the bottom. Standard deviations for ( f ) are 1.2%, 1.6% and 1.5%; for (g) they are 0.4%, 2.0%, 3.9%; and for (h) they are 0.2%, 0.6% and 0.6%, corresponding tocontrol, 10W45 – 59 and 10M46 – 60 ASOs, respectively; n¼ 3.
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Figure 3. Effects of ASOs on PK-M mRNA and protein levels in glioblastoma cells. (a,c) Effects of ASOs 10M46 – 60, 10W45 – 59 and 10MS139 – 153 on endogenous PK-MmRNAs in (a) A172 and (c) U87-MG glioblastoma cells. Radioactive RT-PCR and restriction digest of endogenous PK-M transcripts were performed for the indicatedcell lines 36 h after transfection of 30, 60 or 90 nM 10W45 – 59 or 10M46 – 60 ASO, or 60 or 90 nM 10MS139 – 153 ASO. The control ASO was transfected at 90 nM. %M1,%M2 and %Skp are indicated at the bottom. All s.d. are �4%; n ¼ 3. (b,d ) Immunoblot analysis of PK-M protein isoform levels in (b) A172 and (d ) U87-MG cellstransfected in (a,c). A representative blot from one of three independent experiments is shown. Antibodies used are indicated on the left.
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24 h post-transfection (see the electronic supplementary
material, figure S3a), indicating that the cells were under-
going apoptosis. To confirm and quantitate this
observation, we performed Annexin V staining assays of
A172 and U87-MG cells transfected with ASO 10W45 – 59,
10M46 – 60 or 10MS139 – 153, 36 h after transfection (figure 4a).
As expected, the proportion of Annexin-V-positive cells
increased in an ASO dose-dependent manner, indicating
that ASO-mediated switching of PK-M splicing induces
apoptosis in these cell lines. ASO 10M46 – 60 was the most
potent in inducing apoptosis among the three ASOs tested.
3.8. Antisense oligonucleotide-mediated apoptosis inglioblastoma cells is caused by the downregulationof endogenous PK-M2 expression
ASO transfection elicited a simultaneous increase in PK-M1
expression and a decrease in PK-M2 expression. To pin-
point which of these changes is responsible for inducing
apoptosis in glioblastoma cells, we generated stable cell
lines that express human PK-M1 cDNA in a doxycycline-
inducible manner (figure 4c), or PK-M2 cDNA constitutively
(figure 4e). To investigate the role of PK-M1 in ASO-mediated
apoptosis, we added doxycyline to the PK-M1 inducible cells
for three days, and then treated them with ASO 10W45 – 59,
10M46 – 60 or control ASO. There was a similar increase
in the number of Annexin-V-positive cells in the cells that
did or did not overexpress PK-M1, suggesting that PK-M1
induction did not cause apoptosis in these cells (figure 4d ).
To investigate the role of PK-M2 downregulation in apop-
tosis, we overexpressed PK-M2 in U87-MG and A172 cells,
and then treated them with the maximum dose of ASO
Figure 4. Effects of ASOs on glioblastoma cells. (a,b) Exon 10 ASOs induce apoptosis in glioblastoma cells. (a) U87-MG or A172 cells were transfected with theindicated ASOs at a nominal final concentration of 90 nM, stained with Annexin V-APC/7-AAD 36 h after transfection and analysed by flow cytometry. Thepercentage of Annexin-V-positive cells is indicated for the two right quadrants in each plot. Each plot is a representative of three biological replicates. (b) ASO-induced apoptosis is dose-dependent. The indicated cells were transfected with ASOs 10W45 – 59 or 10M46 – 60 at 30, 60, or 90 nM, or ASO 10MS139 – 153 at 60 or90 nM. The control (Ctl) ASO was transfected at 90 nM. The percentage of Annexin-V-positive cells is plotted for each condition. The identity and dose of ASOs areindicated below the x-axis. Error bars represent s.d. (n ¼ 3). (c,d ) Role of PK-M1 protein isoform in apoptosis induction. (c) Immunoblot analysis of A172 cells stablytransduced with rtTA and doxycycline (dox)-inducible human T7-tagged PK-M1 cDNA. Cells were grown in parallel, with or without doxycycline, and harvested after72 h. Antibodies used are indicated on the left. (d ) Cells were grown as in (c) and then transfected with the indicated ASOs at 60 nM. Cells were then stained andanalysed for Annexin V 36 h after transfection. Histograms indicate the percentage of Annexin-V-positive cells for each condition. Doxcycyline on/off conditions areindicated on the left. Error bars represent s.d. (n ¼ 3). (e,f ) Role of PK-M2 protein isoform in apoptosis induction. A172 or U87 cells were stably transduced withT7-tagged human PK-M2 cDNA; transductants and the parental cell lines were transfected with the indicated ASOs at a nominal final concentration of 90 nM.(e) Immunoblot analysis of cells transfected with the indicated ASOs. Antibodies used are indicated on the left. ( f ) ASO-transfected cells were analysed for AnnexinV as in (d ). Histograms indicate the fold increase in Annexin-V-positive cells, compared with control ASO, for each cell line. Error bars represent s.d. (n ¼ 3).
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expression, but not the induction of PK-M1 expression,
leads to apoptosis in these cell lines.
4. DiscussionWe performed a systematic exon 10 ASO screen, and devel-
oped potent ASOs that increase the inclusion of exon 9 and
skipping of exon 10 in transfected cell lines. We found that
these ASOs switch PK-M splicing especially effectively in
glioblastoma cells, and they induce apoptosis. Finally, we
showed that it is the downregulation of PK-M2 expression
that causes this apoptosis.
Glioblastomas exhibit a high rate of glycolysis, even
under normal oxygen conditions [6]. Consistent with this
phenomenon, the splicing profile of glioblastoma cell lines,
as with other cancer cell lines, is predominantly PK-M2.
Because PK-M2 is a rate-limiting glycolytic enzyme, targeting
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and quantified on a Typhoon 9410 phosphorimager (GE
Healthcare) using MULTI GAUGE software v. 2.3. The percentage
M1 mRNA in endogenous transcripts was calculated using the
GC-content-normalized intensities of the top undigested band
(M1, A), the bottom two digested bands (M2, B1 B2) in the
PstI-digest lanes and the double-skipped species (D), if detect-
able. The percentage M1 mRNA from minigene transcripts
was calculated using the GC-content-normalized intensities of
the top undigested band (a, M1) and other higher-mobility
digested bands corresponding to M2 and its variant species
(b–g, as described above) in the PstI-digest lanes. All the PCR
products were gel-purified, cloned and sequenced to verify
their identities.
5.8. Annexin V AssaysFor Annexin V/7-AAD assays, cells were stained with Annexin
V-APC and 7-AAD, and analysed for apoptosis by flow cyto-
metry using an LSRII Cell Analyzer (Becton–Dickinson).
Briefly, 106 cells were collected 36 h after transfection,
washed twice with phosphate-buffered saline (PBS), resus-
pended in 1 � Binding Buffer (10 mM HEPES, pH 7.4,
140 mM NaCl, 2.5 mM CaCl2) and incubated with 5 ml each
of Annexin V-APC antibody and 7-AAD (Becton–Dickinson)
in the dark for 15 min. Both early-apoptotic (7AAD-/Annexin
Vþ) and late-apoptotic (7AADþ/Annexin Vþ) cells were
included in the quantification.
5.9. ImmunofluorescenceCells were first transfected with ASOs as above, and then plated
onto four-well culture slides (BD Biosciences) 24 h post-trans-
fection. At 36 h post-transfection, cells were washed with PBS
and fixed with 3.7 per cent formaldehyde in PBS for 20 min.
Cells were then permeabilized in 0.1 per cent Triton X-100 in
PBS for 10 min after washing in PBS, blocked for 20 min in
blocking buffer (1% goat serum in PBS) and then incubated
overnight with rabbit monoclonal anti-PKM2 antibody (Cell
Signaling Technology). After washing three times with PBS,
the cells were then incubated for 1 h in blocking buffer contain-
ing Alexa Fluor 594 conjugated goat anti-rabbit secondary
antibody (Molecular Probes/Invitrogen). Cells were washed
with PBS, and then culture slides were disassembled and
mounted with Prolong Gold mounting solution containing
DAPI (Molecular Probes/Invitrogen). Cells were analysed
using a Zeiss Axioplan1 upright fluorescent microscope.
6. AcknowledgementsWe thank members of the Krainer laboratory, in particular
Yimin Hua and Kentaro Sahashi, for helpful discussions.
A.R.K. was supported by NCI grant no. CA13106 and by
the St Giles Foundation. Z.W. is supported by a National
Science Scholarship from the Agency for Science, Technology
and Research, Singapore.
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